Gene therapy is a kind of treatment for genetic diseases and cancers caused by aberration of genes, whose mechanism is to introduce disease-related genes directly to patients in order to normalize the cell function by expressing those genes inside cells. Gene therapy is very effective not only for the treatment of diseases, but also for prevention of many diseases and even more reinforcing the treatment since the therapy can bestow new function on human body by introducing a specific gene.
The crucial point of gene therapy is how introduced genes can be transferred to the nuclei of target cells successfully for mass expression of the genes. After reaching target cells, the introduced genes enter the cells through endocytosis and are expressed in nuclei of the cells. DNA genes can be introduced with liposome, a kind of carrier, because DNA itself cannot pass through cell membrane well. In that case, however, most of the liposome might be destroyed in the middle of transferring into nuclei of the cells, resulting in low transferring efficacy.
Using virus for gene therapy is desirable since foreign genes can be inserted into cells effectively with infectious virus. Particularly, curable genes ought to be inserted in virus DNA by the genetic recombination method and then a great amount of those foreign gene inserted in virus are produced in vitro. By infecting human body with the virus, the curable genes can be transferred into human cells and expressed effectively. Especially, adenovirus can transfer its gene into nuclei of cells, which makes it useful for gene therapy with such effective transmission.
Thymosin β-4, β-10 and β-15 act as major actin monomer-sequestering factors. Thymosin β-4 has 43 amino acids and shares a high degree of homology(85%) at the amino acid level with thymosin β-10. A number of investigations have now suggested that the role of thymosin β-4 and β-10 may be related to mechanisms associated with cell division and/or differentiation. Despite these gene's structural and functional similarities, different expression patterns have been observed. For example, while both thymosins were strongly expressed in fetal brain and other fetal organs, thymosin β-10 levels fell considerably in most adult tissues, and thymosin β-4 expression was down-regulated in metastatic cells of colorectal carcinomas(Hall et al., Mol. Brain Res., 1990, 8:129–135; Hall et al., Mol. Cell. Endocrinol., 1991, 79:37–41; Yamamoto et al., Biochem. Biophys. Res. Commun., 1993, 193:706–710). Another recently discovered member of the β-thymosin family, thymosin β-15, is upregulated in aggressive human prostate cancer (Bao et al., Nat. Med., 1996, 2:1322–28). It is expressed in highly motile, metastatic prostate cancer cells as well as in advanced human prostate and breast cancer (Eadie et al., J. Cell, Biochem., 2000, 77:277–287; Gold et al., Mod. Pathol., 1997, 10:1106–12). Thymosin β-15 differs from other β-thymosins in that its expression correlates with motility and metastasis in highly metastatic prostate carcinoma cells.
Thymosin β-10 is a small actin-binding protein known to sequester actin monomers and thereby induce depolymerization of the intracellular F-actin networks (Nachmias, Curr. Opin. Cell Biol., 1993, 5:56–62; Yu et al., J. Biol. Chem., 1993, 268:502–9; Yu et al., Cell Motil. Cytoskeleton, 1994, 27:13–25). Actin is one of the most abundant structural proteins in the cell (Pollard and Cooper, Ann. Rev. Biochem., 1986, 55:987–1035), and the dynamic equilibrium between monomeric and filamentous actin is shown to be altered in neoplastic/transformed cells (Hall, Ren Fail., 1994, 16:243–54). Alteration of thymosin β-10 expression may thus affect the cellular infrastructure by changing the actin stress fiber, which may further alter the balance of cell growth, cell death, cell attachment and cell migration (Yu et al., J. Biol. Chem., 1993, 268:502–9). During embryogenesis, thymosin β-10 is also highly expressed (Carpintero et al., FEBS Lett., 1996, 394:103–6), which is consistent with constant cell migration and morphogenesis that require cell detachment. Thymosin β-10 was also shown to be involved in inducing processes leading to cell detachment (Iguchi et al., Eur. J. Biochem., 1998, 253:766–770). Thymosin β-10 has also been proposed to have dual functions: programmed cell death and invasion or metastasis (Hall, Cell. Mol. Biol. Res., 1995, 41:167–180; Marian et al., Int. J. Cancer, 1993, 53:278–84).
Differentially expressed genes in normal and cancer cells have recently been identified in order to find novel tumor markers and understand the pathways of cancer development and progression. cDNA microarray is an effective high-throughput method of examining large-scale differential gene expression patterns of specific cDNA populations on a single blot (DeRisi et al., Nat. Genet., 1996, 14:457–60). Fuller et al. successfully used this approach to determine that insulin-like growth factor binding protein 2(IGFBP2) is overexpressed in glioblastoma multiforme (Fuller et al., Cancer Res., 1999, 59:4228–32), and Huang et al. identified superoxide dismutase as a target for the selective killing of cancer cells (Huang et al., Nature, 2000, 407:390–95). An alternative method of gene-expression profiling is the serial analysis of gene expression (SAGE) (Velculescu et al., Science, 1995, 170:484–7; Zhang et al., Science, 1997, 276:1268–72; Hough et al., Cancer Res., 2000, 60:6281–7). An effort to profile gene expression using SAGE was launched by NCBI, and a public database is available for increasing numbers of normal and neoplastic human cell lines and tissues (http://www.sagenetnet.org).
In order to identify proper genes useful for gene therapy for solid malignant tumors, the present inventors searched abnormally expressed genes in solid malignant tumor cells, comparing to normal cell tissues and at last discovered that the expression of thymosin β-10 is decreased remarkably in ovarian cancer cell tissues, compared to normal ovarian cells. And finally, the present inventors have accomplished the present invention by discovering that thymosin β-10 could be used for gene therapy for ovarian cancer, cervical cancer and lung cancer, since thymosin β-10 expressed in adenovirus could suppress the solid malignant tumor cell growth or induce apoptosis of tumor cells.